Certain features of will be down for maintenance Saturday afternoon/evening, February 24th starting at 3:00 pm CT until 9:00 pm CT.

Please note that you still have telephone and email access to our local offices. We apologize for any inconvenience.

Stem Cell Biology

Hematopoietic Stem Cells Protocols

Preparation of Mouse Embryonic Fibroblast (MEF) Feeder Cell Layer
Culture of hES Cells
Induction of hES Cell Differentiation
Expansion of hES-blast Colony (BC) Cells

Preparation of Mouse Embryonic Fibroblast (MEF) Feeder Cell Layer

  • Eviscerate 12.5 dpc ICR or CD-1 mouse embryos leaving the heads on; rinse with PBS (P7059) and mince with scissors for approximately 3–5 minutes in a dry 60 mm Petri dish.
  • Add 5 ml of 37 °C trypsin (T5266) per mouse, constantly pipetting with a 5 ml tissue culture pipette for 5 minutes; transfer to a 50 ml tube with 20 ml of MEF growth medium (DMEM medium (D5796) supplemented with 10% FBS (F6178), GlutaMax, Penicillin and streptomycin (G6784) and centrifuge at approximately 1000 rpm for 5 minutes.
  • Remove the supernatant and resuspend the pellet in MEF growth medium.
  • Plate the suspension onto gelatin-coated plates at a density of 1.5 embryos per 150 mm plate (P0).
  • Expand MEFs once (1:5 split) usually within 1–2 days and freeze (P1).
  • Add 10 µg/ml Mitomycin C (M4287) to the media of a confluent plate of MEFs and incubate at 37 °C for 3 hours.
  • Harvest by trypsinization and plate in MEF growth medium at a density of 50–60 thousand cells/cm2.
  • Pre-treat plate with 0.1% gelatin (G1393) for at least 4 hours, then plate 7.5 X 105 cells per well of six-well plate. Incubate at 37 °C with 5% CO2 for at least 24 hours. The MEF feeder layer is now ready for plating hES cells.


Culture of hES Cells

  • Add 0.5 ml 0.05% trypsin-0.53 mM EDTA (T4049) to one well (hES cells) of six-well plate, incubate at 37 °C for 3–4 min, then pipette to produce smaller cell clumps (2–5 cells).
  • Collect cells by adding 3 ml of hES cell growth medium (KO-DMEM medium with 10% Plasmanate, 10% Knockout Serum Replacement (S0638), 10 ng/ml of human LIF (L5283), 8 ng/ml of human bFGF (F0291). 1X non-essential amino acid (M7145), 1X Glutamax-I, and
    1X β-mercaptoethanol (M7522) and spin down for 4 min at 1000 rpm.
  • Resuspend cells in 9 ml of hES cell growth medium and re-plate in 3 wells of six-well plate with pre-formed MEF feeder.
  • Change half (1.5 ml) of the medium every 24 hours until the cells reach 70–80% confluence.


Induction of hES Cell Differentiation

  • Collect hES cells as above, and count cell numbers. Usually one well of 80% confluent, undifferentiated hES cells will generate approximately 2 million cells.
  • Resuspend cells from one well in 3 ml Stemline II medium (S0192) supplemented with 50 ng/ml of VEGF (V4512) and 50 ng/ml of BMP-4 (B2680), plate in one well of six-well ultra low plate, and incubate at 37 °C with 5% CO2. EBs will be formed in the first 24 hours.
  • Replace half the medium (1.5 ml) with fresh Stemline II medium (S0192) supplemented with 50 ng/ml of VEGF (V4512), 50ng/ml of BMP-4 (B2680), 40ng/ml of SCF (S7901), 40ng/ml of Tpo (T4184) and 40ng/ml of FLT3 (F3422) ligand, and continue incubation.
  • Collect EBs after 84 hours and the dissociate EBs by 0.05% trypsin-0.53 mM EDTA for 2–5 min. Prepare single cell suspension by passing through 22G needle 3–5 times and a 40 µm strainer.
  • Count cell numbers and resuspend cells in Stemline II medium at 2–5 X 106 cells/ml.


Expansion of hES-blast Colony (BC) Cells

  • Mix single cell suspension (0.1 ml, 2 to 5 X 105 cells) with 2–3 ml of BC growth medium (BGM, 1.0% methylcellulose in Isocve’s MDM (I6529), 1–2% bovine serumalbumin (A9418), 0.1 mM
    2-mercaptoethanol (M7522), 10 µg/ml rh-insulin, 200 µg/ml iron saturated human transferring (T0665) 20 ng/ml rh-GM-CSF (G0282), 20ng/ml rh-IL-3 (I1646), 20 ng/ml rh-IL-6 (I3268), 20 ng/ml rh-G-CSF (G8160), 3 to 6 units/ml rh-Epo (E5627), 50ng/ml rh-SCF (S7901), 50 ng/ml rh-VEGF (V7259), 50 ng/ml rh-BMP-4 (B2680), 50 ng/ml of Tpo (T1568) and FL) with brief vortex, and stand for 5 min.
  • Transfer the BGM-cell mixture to one well of six-well ultra low plate by using a syringe (3 ml) attached with a 16G needle, and incubate at 37 °C with 5% CO2.
  • Usually BCs are visible at 3 days, and after 4–6 days, grape-like hES-BCs can be easily identified under microscopy. After 6–7 days, BCs can be picked up using a mouth-glass capillary.


Protocols taken in part from:

  • Lu, S. J. et al. Generation of functional hemangioblasts from human embryonic stem cells. Nature Methods 2007, 418, 501–509.


back to top